Tian Guangming, Wang Qin, Wei Xuetuan, Ma Xin, Chen Shouwen
School of Animal Science, Yangtze University, Jingzhou 434025, People's Republic of China; Hubei Key Laboratory of Waterlogging Disaster and Agriculture Use of Wetland, Yangtze University, Jingzhou 434025, People's Republic of China.
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan 430062, People's Republic of China.
Enzyme Microb Technol. 2017 Apr;99:9-15. doi: 10.1016/j.enzmictec.2017.01.002. Epub 2017 Jan 6.
Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis.
聚γ-谷氨酸（γ-PGA）是一种天然生物聚合物，由于其具有保水、阳离子螯合、无毒和可生物降解等特性，被广泛应用于化妆品、医药、食品、水处理和农业等领域。细胞内谷氨酸作为γ-PGA的底物，是γ-PGA高产的限制因素。枯草芽孢杆菌和地衣芽孢杆菌都是重要的γ-PGA生产菌株，枯草芽孢杆菌利用独特的谷氨酰胺合成酶/谷氨酸合酶（GOGAT/GS）途径在体内合成谷氨酸。然而，关于地衣芽孢杆菌中谷氨酸合成途径的了解却很少。本研究旨在通过体内和体外实验，对地衣芽孢杆菌谷氨酸合成中的谷氨酸脱氢酶（RocG）进行表征。通过重新引导碳通量分布，rocG基因缺失突变体WX-02ΔrocG产生的细胞内谷氨酸浓度为90ng/log（CFU），仅为野生型WX-02（380ng/log（CFU））的23.7%。此外，突变体WX-02ΔrocG的γ-PGA产量为5.37g/L，与野生型（9.82g/L）相比降低了45.3%。对RocG的体外酶活性测定表明，RocG对2-氧代戊二酸的亲和力高于谷氨酸，且谷氨酸合成速率远高于降解速率。这可能是首次揭示地衣芽孢杆菌谷氨酸合成途径及RocG具体功能的研究。结果表明，通过改善细胞内谷氨酸合成可以提高γ-PGA产量。
